Without limiting the scope of the invention, its background is described in connection with novel compositions and methods for enhancement of skeletal fracture healing, in the tissue engineering, and for therapeutic hair regeneration using Developmentally Regulated Endothelial Locus 1 (Del-1) genes and proteins as essential growth factors.
Del-1 is a secreted extracellular matrix protein that has been shown to be an angiogenic factor. Del-1 protein expressed in a recombinant baculovirus system was shown to promote αvβ3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del-1 was associated with clustering of the integrin αvβ3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes followed by downstream kinase signaling. When recombinant Del-1 was evaluated in an in ovo chick chorioallantoic membrane (CAM) assay, it was found to have potent angiogenic activity. (Penta K, et al. J Biol Chem. 274 (16) (1999) 11101-9). Importantly, neutralizing antibody to Del-1 or mutation of the RGD motif will inhibit this angiogenic activity. Thus, Del-1 is a secreted extracellular matrix protein that is capable of stimulating angiogenesis through integrin binding.
The full length human and murine Del-1 protein of 480 amino acids has three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. (Hidai C, et al. Genes Dev. 12(1) (1998) 21-33). During embryogenesis, Del-1 is spatiotemporally expressed during embryogenesis prominently in the developing vasculature, portions of the brain, and in cartilaginous structures. In the early Hidai study, expression of Del-1 was also noted in endochondral bone of 9.5 day mouse embryos as well as the hypertropic chondrocytes of limb bones and vertebral bodies. By 13.5 days, Del-1 expression in endothelial type cells ceased although expression of Del-1 in hypertropic chondrocytes was retained. Although considered possible that Del-1 was directly involved in supporting bone function, it was thought to be a more attractive hypothesis that expression of Del-1 by chondrocytes reflected a mechanism by which these cells regulate vascularization of bone-forming regions. No Del-1 was found to be expressed in adult tissues.
Subsequently, Del-1 was found to be present in adult articular cartilage in the cell-associated matrix of freshly isolated superficial chondrocytes and was thought to interact with integrin αvβ3, which is present in the superficial layer of articular cartilage. (Pfister, et al. Biochemical and Biophysical Research Communications 286 (2001) 268).
Bone fracture is a very common wound experienced by virtually all persons at some time in their lives. It is estimated that 5-10% of all fractures show impaired healing, leading to delayed or non-union. Thus, chemical or physical methods to accelerate bone healing are of great interest. As with soft tissue wound healing, fracture healing progresses through three general stages: inflammation, proliferation, and remodeling. However, because of supporting strength needed in bone, fractures generally take longer to heal than soft tissue wounds. New bone forms through a cartilaginous intermediate, which can be seen by x-ray about 10 days after fracture. The cartilage is soft and flexible and takes weeks to months for replacement with hard bony tissue. Weight supporting long bones, such as the femur, can take 3-5 months to heal. Healing requires immobilization of the fracture and is associated with considerable morbidity.
Growth factors have been studied in an effort to augment fracture healing with various results. The growth factors IGF-1 and TGF-β1 are known to stimulate fracture healing including through an earlier appearance of cartilage and an enhanced maturation of the callus tissue. (Wildmann B et al. J Biomed Mater Res B Appl Biomater 65(1)(2003) 150-6). Osteosynthetic implants including growth factors have yielded some encouraging results in animal studies. Thus, implants composed of poly(D,L-lactide) (PDLLA) impregnated with insulin-like growth factor-1 (IGF-1) have been reported to accelerate fracture healing significantly. (Schmidmaier et al. Bone 28(4) (2001) 341-50). Likewise, a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model resulted in biomechanical strength of treated motion segments that was not statistically different from an autograft suggesting an effective alternative to autograft for bone grafting procedures. (Spiro et al. Anat Rec 263(4) (2001) 388-95).
Bone morphogenetic protein-2 (BMP-2) has been reported to increase the rate of callous formation without affecting the amounts of bone or cartilage ultimately produced. (Bax BE et al. Calcif Tissue Int 65(1) (1999) 83-9). In a large animal study, injection of osteogenic protein-1 (BMP-7) into the fracture gap was associated with higher stiffness and strength 2 weeks after injection. (Blokhuis T J, et al. Biomaterials 22(7) (2001) 725-30). A human randomized, controlled, single-blind clinical trial in open tibial shaft fractures has been conducted in which a recombinant human BMP-2 implant (rhBMP-2 applied to an absorbable collagen sponge) was placed over the fracture at the time of definitive wound closure. Use of rhBMP-2, albeit at large doses, was significantly superior to control in reducing the frequency of secondary interventions and overall invasiveness of the procedures, accelerating fracture and wound-healing, and reducing the infection rate in patients with an open fracture of the tibia. (Govender S et al. J Bone Joint Surg Am 84-A(12) (2002) 2123-34). Similarly, a human anterior lumbar fusion clinical trial has comparing rhBMP-2 on an absorbable collagen sponge (INFUSE® Bone Graft) with use of an autograft transferred from the iliac crest implanted in a fusion device. The patients treated with rhBMP-2 had statistically superior outcomes with regard to length of surgery, blood loss, hospital stay, re-operation rate, median time to return to work, and fusion rates at 6, 12, and 24 months. Burkus J K et al. J Spinal Disord Tech 16(2) (2003) 113-22. In 2004, the FDA granted pre-market approval P000054 for use of the INFUSE® rhBMP-2 collagen sponge in treating acute, open tibial shaft fractures that have been stabilized with intermedullary nail fixation. However, the FDA new device approval overview notes that use of the INFUSE® device caused fractures to heal in a similar manner to bones not treated with the device. Patients who received INFUSE® required fewer interventions to promote healing compared to patients who did not receive the device, however, patients who received the device and required an intervention healed at a slower rate compared to patients who did not receive the device.
The utility of TGF-beta in bone healing has been conflicting (Tielinen et al. Arch Orthop Trauma Surg 121(4) (2001) 191-6). Fibroblast growth factor (FGF) has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing (Nakajima et al. J Orthop Res 19(5) (2001) 935-44). There remains a need for further growth factors that are able to accelerate the rate of events in early fracture healing including bone grafting.
Tissue disease and organ failure leads to an estimated 8 million surgical procedures annually in the United States. Treatments in the form of transplantation and tissue reconstruction are among the most expensive, costing billions of dollars a year. Tissue engineering using biodegradable scaffolds impregnated with growth factors or autologous cells that are able to populate the scaffold is a promising technique for the generation of replacement cartilaginous tissues including nasal septum, ear, throat, and the cartilage lining the joints (“articular cartilage”).
A major problem faced by the aging population is osteoarthritis (OA). See Felson D T. Clinical practice. Osteoarthritis of the knee. N Engl J Med 354(8) (2006) 841-8. Cartilage serves as a cushion for the impact of locomotion and excessive wear at joint surfaces leads to loss of the articular cartilage with ensuing inflammation and pain. The only reliable method to ease pain in these patients is a total joint replacement, a major procedure with significant risks for morbidity and mortality. Cartilage has a very limited ability to regenerate over time so there is no appreciable replacement of cartilage lost to OA. Cartilage has limited capacity for self-repair due in part to a poor blood supply. Articular cartilage, is particularly difficult to repair due to an isolated chondrocyte (cartilage-producing cell) microenvironment, as well as high forces generated in the joint. Over the long term, defects may progress to end stage arthritis, leading to the need for joint replacement. A popular dietary supplement that has been purported to aid OA is glucosamine and chondroitin sulfate. A recent study has just demonstrated that there is no significant benefit of these supplements in chronic disease, but the volume of sales of the supplements suggests the extent of this disease and the numbers of people seeking relief from pain related to it. See Clegg D O, Reda D J, Harris C L, et al. Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med 2006; 354(8):795-808.
One solution to repair of cartilaginous tissue is to administer autologous chondrocytes in combination with novel synthetic scaffolds to provide immediate structural repair as well as a new population of cells capable of growing new cartilaginous tissue. There is currently one approved tissue engineered cartilage that is used for this purpose (Carticel, Genzyme, Cambridge, Mass.). This therapy has suffered from several problems. The patient must not only endure the harvesting of autologous chondrocyte donor tissue, but must wait for proliferation of these cells in vitro prior to implantation. There is potential morbidity from the donor site required to acquire autologous chondrocytes, weeks are required for expansion of to create a small construct, many of these grafts disintegrate after implantation, and finally, the costs of this therapy are extremely high. What is needed is a factor able to enhance the in vitro populating of scaffolds or, alternatively, the recruitment and growth potentiation of chondrocytes to the scaffold in vivo by impregnating the scaffold with a chondrocyte growth factor. What is further needed is a more readily available source of donor cells.
Another approach to managing OA is to prevent early disease from worsening. A concept that has been emerging over the past decade has been the role of apoptosis in the pathogenesis of OA. It has been well documented that articular injury through trauma or chronic impaction leads to death of the articular cartilage. More recently, multiple studies have shown increased rates of apoptosis in joints that have suffered trauma. These studies have suggested that affecting chondrocyte apoptosis may be a method to preventing development of OA following injury to the articular cartilage or the progression of OA after it has initially manifested, but before the development of severe disease. What is need is an inhibitor of chondrocyte apoptosis such that loss of cartilage can be controlled.
Another therapeutic need is for a viable hair regrowth compositions and methods. Hair loss on the scalp can be a consequence of aging, hormonal changes, exposure to certain drugs, and/or a family history of baldness. Hair replacement surgery, which is the only permanent hair replacement option, requires either invasive skin flap surgery or autologous grafts. The only available medical therapies are with the topical drug minoxidil (brand name Rogaine®) or the oral medication finasteride (brand name Propecia®), each of which take up to 6 months of treatment before it can be apparent whether the drugs will work. Further therapies for hair regrowth are needed.